1. Field of the Invention
The present invention relates to a novel GMP synthetase and a DNA that codes for it. A microorganism that has GMP synthetase and its enzymatic activity can be used for the production of nucleic acid compounds such as guanosine 5xe2x80x2-monophosphate, which is useful as a raw material of seasonings and so forth.
2. Description of the Related Art
In the conventional production of nucleic acid compounds such as guanosine 5xe2x80x2-monophosphate (also called 5xe2x80x2-guanylic acid, hereinafter abbreviated as xe2x80x9cGMPxe2x80x9d) and guanosine by fermentation, mutant strains that are imparted with adenine auxotrophy and nucleic acid analogue resistance have been utilized in a medium containing a limited amount of adenine compounds (Japanese Patent Publication (Kokoku) Nos. 55-2956/1980 and 55-45199/1980).
Usual mutagenesis treatments often introduce mutations in genes of resulting mutant strains other than a target gene. In addition, because complicated controlling mechanisms are present in the biosynthetic pathways of nucleic acid compounds, it is difficult to obtain a microorganism that produces a marked amount of a certain nucleic acid compound. Therefore, mutant strains obtained by conventional methods for breeding bacterial strains have not necessarily been satisfactory strains.
GMP is synthesized by an amination reaction catalyzed by GMP synthetase and utilizing L-glutamine as an amino group donor from 5xe2x80x2-inosinic acid (IMP) via xanthylic acid (XMP). Therefore, there has been proposed a method for producing GMP which utilizes Escherichia coli transformed with a GMP synthetase gene (guaA) of Escherichia coli (Japanese Patent Publication (Kokoku) No. 7-16431/1995). The guaA gene has been isolated from Bacillus subtilis (J. Bacteriol., 174, 1883-1890 (1992), EMBL/GenBank/DDBJ Accession M83691), Corynebacterium ammoniagenes (Korean Patent Publication No. 96-7743, EMBL/GenBank/DDBJ Accession Y10499), Borrelia burgdorferi (J. Bacteriol., 176, 6427-6432 (1994), EMBL/GenBank/DDBJ Accession L25883), Dictyostelium discoideum (J. Biol. Chem., 266, 16448-16452 (1991), EMBL/GenBank/DDBJ Accession M64282) and so forth.
However, no guaA gene of Brevibacterium lactotermentum (Corynebacterium glutamicum) has been known.
An object of the present invention is to provide a guaA gene of Brevibacterium lactofermentum (Corynebacterium glutamicum) and a transformant introduced with the gene.
The inventors of the present invention actively studied in order to achieve the aforementioned object. As a result, they found a plurality of regions conserved in amino acid sequences deduced from the known guaA genes, and based on the amino acid sequences of these regions, selected a region and a sequence suitable for amplifying a guaA gene of Brevibacterium lactofermentum (Corynebacterium glutamicum) by PCR. Then, they successfully isolated a part of the guaA gene by utilizing the selected sequence, and further obtained the guaA gene in its full length. Thus, they accomplished the present invention.
That is, the present invention provides the followings.
(1) A protein defined in the following (A) or (B):
(A) a protein which has the amino acid sequence of SEQ ID NO: 2, or
(B) a protein which has the amino acid sequence of SEQ ID NO: 2 including substitution, deletion, insertion, addition or inversion of one or several amino acids, and has GMP synthetase activity.
(2) A DNA which codes for a protein defined in the following (A) or (B):
(A) a protein which has the amino acid sequence of SEQ ID NO: 2, or
(B) a protein which has the amino acid sequence of SEQ ID NO: 2 including substitution, deletion, insertion, addition or inversion of one or several amino acids, and has GMP synthetase activity.
(3) The DNA according to (2), which is a DNA defined in the following (a) or (b):
(a) a DNA which has the nucleotide sequence comprising at least the nucleotide sequence of the nucleotide numbers 887 to 2455 in SEQ ID NO: 1; or
(b) a DNA which is hybridizable with a nucleotide sequence comprising at least the nucleotide sequence of the nucleotide numbers 887 to 2455 in SEQ ID NO: 1 under a stringent condition, and codes for a protein having GMP synthetase activity.
(4) The DNA according to (3), wherein the stringent condition means a condition that washing is performed at 1xc3x97SSC, 0.1% SDS and 60xc2x0 C.
(5) A transformant which is introduced with the DNA according to any one of (2) to (4) in a form that allows expression of a protein encoded by the DNA.
The present invention will be explained in detail hereafter.
The DNA of the present invention is a DNA that codes for GMP synthetase derived from Brevibacterium lactofermentum (Corynebacterium glutamicum). Brevibacterium lactofermentum has now been unified into Corynebacterium glutamicum, but it may be referred to as Brevibacterium lactofermentum in the present specification for convenience.
A part of the DNA of the present invention can be obtained from chromosomal DNA of Brevibacterium lactofermentum by PCR. Primers used for the PCR can be designed as follows. That is, an amino acid sequence of a region conserved in GMP synthetases can be retrieved by alignment of amino acid sequences of the GMP synthetases deduced from nucleotide sequences of the guaA genes which have been cloned from several microorganisms, for example, Escherichia coli, Bacillus subtilis, Corynebacterium ammoniagenes, Borrelia burgdorferi, Dictyostelium discoideum and so forth. Then, the primers can be designed based on the retrieved conserved region. In this procedure, the PCR primers are designed by selecting such a region that the number of corresponding codons should become as few as possible, and referring to occurring frequency of the codons in Brevibacterium lactofermentum. 
Chromosomal DNA of B. lactofermentum can be prepared by, for example, the method of Saito and Miura (Biochem. Biophys. Acta., 72, 619, (1963)), and the method of K. S. Kirby (Biochem. J., 64, 405, (1956)) or the like.
As preferred primers and PCR condition for amplification of the guaA gene, the combinations of primers and the reaction conditions used for Reactions 2, 3 and 4 shown in Table 1 can be mentioned. By using these reaction conditions, reaction products having sizes of about 0.5 kbp, about 1.2 kbp and about 0.7 kbp, respectively, can be obtained. Among these reaction products, the fragment of about 1.2 kbp overlaps the fragment of about 0.5 kbp.
Based on a partial sequence of the guaA gene obtained as described above, upstream and downstream flanking regions of the partial sequence can be obtained by the cassette-ligation mediated PCR method (Molecular and Cellular Probes, 6, 467-475) or the like, and thus a full length guaA gene can be obtained. That is, a region flanking a known region on chromosomal DNA can be obtained by ligating a cassette to a chromosomal DNA fragment digested with a suitable restriction enzyme, and amplifying it by PCR utilizing a primer corresponding to the known region and a primer corresponding to the cassette. In this procedure, by using a dephosphorylated 5xe2x80x2 end of the cassette, a nick is produced at the ligation site of the chromosomal DNA fragment and the 5xe2x80x2 end of the cassette. Therefore, the DNA synthesis started from the cassette primer is stopped at this ligation site, and thus only DNA synthesized from the synthetic primer serves as a template for the synthesis from the cassette primer to form a complementary strand. The nucleotide sequence of the obtained amplification product is determined, then specific primers for the newly determined nucleotide sequence are synthesized, and PCR is performed by using the chromosomal DNA fragment similarly ligated to the cassette as a template. By repeating this procedure, the upstream and downstream flanking regions can be obtained. A kit utilizing this method is commercially available (TAKARA LA PCR in vitro Cloning Kit, Takara Shuzo), and it can be utilized for obtaining the DNA of the present invention.
Since the nucleotide sequences of the DNA of the present invention and the flanking regions have been revealed, the DNA of the present invention can be directly amplified by PCR utilizing oligonucleotides synthesized based on the nucleotide sequences as primers and chromosomal DNA of coryneform bacteria as a template. As such primers, oligonucleotides which have the nucleotide sequences shown in SEQ ID NO: 12 and SEQ ID NO: 13 can be mentioned. In a DNA obtained by using these primers, the SD (Shine-Dalgarno) sequence is added upstream of the region coding for GMP synthetase, and restriction enzyme recognition sites are added to the both ends. Moreover, the DNA of the present invention can also be isolated from a chromosomal DNA library by hybridization utilizing an oligonucleotide synthesized based on its nucleotide sequence as a probe.
An example of nucleotide sequence of a DNA fragment containing the DNA of the present invention, which can be obtained as described above, is shown in SEQ ID NO: 1 of Sequence Listing. In this nucleotide sequence, the region consisting of nucleotides of nucleotide numbers 887-2455 codes for GMP synthetase, which is the protein of the present invention.
The DNA of the present invention may code for GMP synthetase including substitution, deletion, insertion, addition, or inversion of one or several amino acids at one or a plurality of positions, provided that the activity of GMP synthetase encoded thereby is not deteriorated. The number of xe2x80x9cseveralxe2x80x9d amino acids differs depending on the position or the type of amino acid residues in the three-dimensional structure of the protein. This is because of the following reason. That is, some amino acids such as isoleucine and valine are amino acids having high homology to one another. The difference in such an amino acid does not greatly affect the three-dimensional structure of the protein. Therefore, the protein encoded by the DNA of the present invention may be one which has homology of not less than 80%, preferably 90% with respect to the entire amino acid residues for constituting. GMP synthetase, and which has the GMP synthetase activity. More appropriately, the number of xe2x80x9cseveralxe2x80x9d amino acids is 2 to 104, preferably 2 to 50, and more preferably 2 to 10.
Alternatively, DNA of the present invention may be DNA which has homology of not less than 60%, preferably not less than 70% and more preferably not less than 80% with the region consisting of nucleotides of nucleotide numbers 887-2455 in SEQ ID NO: 1 in nucleotide sequence level, provided that the protein encoded by the DNA has GMP synthetase activity.
DNA, which codes for the substantially same protein as GMP synthetase as described above, is obtained, for example, by modifying the nucleotide sequence, for example, by means of the site-directed mutagenesis method so that one or more amino acid residues at a specified site involve substitution, deletion, insertion, addition, or inversion. DNA modified as described above may be obtained by the conventionally known mutation treatment. The mutation treatment includes a method for treating DNA coding for GMP synthetase in vitro, for example, with hydroxylamine, and a method for treating a microorganism, for example, a bacterium belonging to the genus Escherichia harboring DNA coding for GMP synthetase with ultraviolet irradiation or a mutating agent such as N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine (NTG) and nitrous acid usually used for the mutation treatment.
The substitution, deletion, insertion, addition, or inversion of nucleotide as described above also includes mutant or variant which naturally occurs, for example, on the basis of the individual difference or the difference in species or genus of the microorganism which harbors GMP synthetase
The DNA, which codes for substantially the same protein as GMP synthetase, is obtained by isolating DNA which is hybridizable with DNA having, for example, a nucleotide sequence corresponding to nucleotide numbers of 887 to 2455 of the nucleotide sequence depicted in SEQ ID NO: 1 in Sequence Listing under a stringent condition, and which codes for a protein having the GMP synthetase activity, from DNA coding for GMP synthetase having mutation or from a cell harboring it. The xe2x80x9cstringent conditionxe2x80x9d referred to herein is a condition under which so-called specific hybrid is formed, and non-specific hybrid is not formed. It is difficult to clearly express this condition by using any numerical value. However, for example, the stringent condition includes a condition under which DNA""s having high homology, for example, DNA""s having homology of not less than 60% are hybridized with each other, and DNA""s having homology lower than the above are not hybridized with each other. Alternatively, the stringent condition is exemplified by a condition in which washing in hybridization procedure is performed at 1xc3x97SSC, 0.1% SDS, 60xc2x0 C., preferably at 1xc3x97SSC, 65xc2x0 C., and more preferably at 0.5xc3x97SSC, 65xc2x0 C.
The gene, which is hybridizable under the condition as described above, includes those having a stop codon generated in a coding region of the gene, and those having no activity due to mutation of active center. However, such mutants can be easily removed by ligating the gene with a commercially available activity expression vector, and measuring the GMP synthetase activity in accordance with the method described above.
The protein of the present invention is a protein encoded by the aforementioned DNA of the present invention, and has the amino acid sequence shown in SEQ ID NO: 2. The protein of the present invention may have an amino acid sequence of SEQ ID NO: 2 including substitution, deletion, insertion, addition or inversion of one or several amino acids, so long as it has GMP synthetase activity.
The GMP synthetase of the present invention has an activity for catalyzing the reaction that generates GMP from XMP. In this reaction, it can utilize L-glutamine and (NH4)2SO4 as an amino group donor. The measured Km values for L-glutamine and ammonia of the GMP synthetase of the present invention were about 9.6 mM and 8.2 mM, respectively.
Moreover, when L-glutamine was used as an amino group donor, the GMP synthetase of the present invention showed the maximum activity around pH 6.5. When ammonia was used as the amino group donor with a constant amount of (NH4)2SO4, which was added as a source of the amino group donor, it showed the maximum activity around pH 8.0 to 9.0. The GMP synthetase of the present invention was stable for at least 15 minutes at up to about 23xc2x0 C.
In a transformant which is introduced with the DNA of the present invention in a form that allows expression of the GMP synthetase encoded by the DNA, the GMP synthetase activity is enhanced compared with a cell that is not transformed with it.
The xe2x80x9ccoryneform bacteriaxe2x80x9d includes bacteria having been hitherto classified into the genus Brevibacterium but united into the genus Corynebacterium at present (Int. J. Syst. Bacteriol., 41, 255 (1981)), and include bacteria belonging to the genus Brevibacterium closely relative to the genus Corynebacterium. Examples of such coryneform bacteria include the followings.
Corynebacterium acetoacidophilum 
Corynebacterium acetoglutamicum 
Corynebacterium alkanolyticum 
Corynebacterium callunae 
Corynebacterium glutamicum 
Corynebacterium lilium (Corynebacterium glutamicum)
Corynebacterium melassecola 
Corynebacterium thermoaminogenes 
Corynebacterium herculis 
Brevibacterium divaricatum (Corynebacterium glutamicum)
Brevibacterium flavum (Corynebacterium glutamicum)
Brevibacterium immariophilum 
Brevibacterium lactofermentum (Corynebacterium glutamicum)
Brevibacterium roseum 
Brevibacterium saccharolyticum 
Brevibacterium thiogenitalis 
Brevibacterium ammoniagenes (Corynebacterium ammoniagenes)
Brevibacterium album 
Brevibacterium cerinum 
Microbacterium ammoniaphilum 
Specifically, the following strains of these bacteria are exemplified:
Corynebacterium acetoacidophilum ATCC13870
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium alkanolyticum ATCC21511
Corynebacterium callunae ATCC15991
Corynebacterium glutamicum ATCC13020, 13032, 13060
Corynebacterium lilium (Corynebacterium glutamicum) ATCC15990
Corynebacterium melassecola ATCC17965
Corynebacterium thermoaminogenes AJ12340 FERM BP-1539)
Corynebacterium herculis ATCC13868
Brevibacterium divaricatum (Corynebacterium glutamicum) ATCC14020
Brevibacterium flavum (Corynebacterium glutamicum) ATCC13826, ATCC14067
Brevibacterium immariophilum ATCC14068
Brevibacterium lactofermentum (Corynebacterium glutamicum) ATCC13665, ATCC13869
Brevibacterium roseum ATCC13825
Brevibacterium saccharolyticum ATCC14066
Brevibacterium thiogenitalis ATCC19240
Corynebacterium ammoniagenes (Brevibacterium ammoniagenes) ATCC6871
Brevibacterium album ATCC15111
Brevibacterium cerinum ATCC15112
Microbacterium ammoniaphilum ATCC15354
These strains can be provided from, for example, the American Type Culture Collection. Each strain is assigned its registration number, and one can request provision of each strain by referring to its registration number. The registration numbers corresponding to the strains are indicated on the catalog of the American Type Culture Collection (Address: 12301 Parklawn Drive, Rockville, Md. 20852, United States of America). The AJ12340 strain was deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (zip code: 305-8566, 1-3 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan) under the provisions of the Budapest Treaty, and received an accession number of FERM BP-1539.
The DNA of the present invention is preferably ligated to a vector DNA autonomously replicable in a cell of Escherichia coli and/or coryneform bacteria to form a recombinant DNA. By introducing this recombinant DNA into an Escherichia coli cell, the subsequent procedure can be made easy. The vector autonomously replicable in Escherichia coli cells is preferably a plasmid vector autonomously replicable in the host cell, and examples thereof include pUC19, pUC18, pBR322, pHSG299, pHSG399, pHSG398, RSF1010 and so forth.
As the vector autonomously replicable in coryneform bacterium cells, there can be mentioned pAM330 (refer to Japanese Patent Laid-open (Kokai) No. 58-67699/1983), pHM1519 (refer to Japanese Patent Laid-open (Kokai) No. 58-77895/1983) and so forth. Moreover, if a DNA fragment having an ability to make a plasmid autonomously replicable in coryneform bacteria is taken out from these vectors and inserted into the aforementioned vectors for Escherichia coli, they can be used as a so-called shuttle vector autonomously replicable in both of Escherichia coli and coryneform bacteria. Examples of such a shuttle vector include those mentioned below. There are also indicated microorganisms which harbors each vector, and accession numbers thereof at the international depository are shown in the parentheses, respectively.
pAJ655 Escherichia coli AJ11882 (FERM BP-136)
Corynebacterium glutamicum SR8201 (ATCC39135)
pAJ1844 Escherichia coli AJ11883 (FERM BP-137)
Corynebacterium glutamicum SR8202 (ATCC39136)
pAJ611 Escherichia coli AJ11884 (FERM BP-138)
pAJ3148 Corynebacterium glutamicum SR8203 (ATCC39137)
pAJ440 Bacillus subtilis AJ11901 (FERM BP-140)
pHC4 Escherichia coli AJ12617 (FERM BP-3532)
In order to prepare recombinant DNA by ligating the gene coding for GMP synthetase and a vector which can function in a cell of coryneform bacterium, the vector is digested by restriction enzyme(s) corresponding to the termini of the gene coding for GMP synthetase. Ligation is generally performed by using a ligase such as T4 DNA ligase.
To introduce the recombinant DNA prepared as described above to a coryneform bacterium, any known transformation methods can be employed. For instance, employable are a method of treating recipient cells with calcium chloride so as to increase the permeability of DNA, which has been reported for Escherichia coli K-12 [see Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)]; and a method of preparing competent cells from cells which are at the growth phase followed by introducing the DNA thereinto, which has been reported for Bacillus subtilis [see Duncan, C. H., Wilson, G. A. and Young, F. E., Gene, 1, 153 (1977)]. In addition to these, also employable is a method of making DNA-recipient cells into the protoplast or spheroplast which can easily take up recombinant DNAs followed by introducing the recombinant DNA into the cells, which is known to be applicable to Bacillus subtilis, actinomycetes and yeasts [see Chang, S. and Choen, S. N., Molec. Gen. Genet., 168, 111 (1979); Bibb, M. J., Ward, J. M. and Hopwood, O. A., Nature, 274, 398 (1978); Hinnen, A., Hicks, J. B. and Fink, G. R., Proc. Natl. Sci., USA, 75, 1929 (1978)]. The electric pulse method can be also applied (refer to Japanese Patent Publication Laid-Open No. 2-207791).
A microorganism that is transformed with the DNA of the present invention can be utilized for the production of GMP synthetase. Moreover, since a microorganism, for example Brevibacterium lactofermentum, transformed with the DNA of the present invention and has enhanced GMP synthetase activity is excellent in the ability to generate GMP from XMP, it can be used for the production of GMP by fermentation or enzymatic process utilizing bacterial cells.
According to the present invention, there is provided a GMP synthetase derived from Brevibacterium lactofermentum and a gene coding for it. The GMP synthetase can be used for the production of GMP. A transformant that harbors the gene can be used for the production of GMP synthetase or GMP.